Abstract: Acute myeloid leukemia (AML) is the most common adult leukemia with ~20,000 new cases expected in 2016 in the US and a 5-year survival rate of only 25%. The primary cause of death for AML is due to disease relapse. The consequences of relapse are significant with it not atypical to see a relapse rate of 60% of which only 11% survive after 5 years. If clinicians could easily identify a patient?s minimum residual disease (MRD) before the tumor rapidly expands to florid to relapse, preemptive therapies could be taken with better patient outcomes. Unlike other leukemias, AML?s inter-patient heterogeneity is immense; there is no characteristic genetic mutation or aberrant protein expression pattern for all AML patients, thus complicating the broad applicability of conventional MRD approaches: PCR, next generation sequencing or multi- parameter flow cytometry. If MRD could be detected in all AML patients, from peripheral blood (not bone marrow), with higher sensitivity than achieved by current methods, the corresponding assay could assist in guiding therapy to enable precision medicine resulting in better patient outcome. In this fast-tracked Phase II application, we propose to build upon a highly successful project to commercialize a microfluidic assay which is highly sensitive for MRD testing, permits frequent sampling by using peripheral blood as opposed to a bone marrow biopsy and is currently applicable to ~90% of all AML patients. The assay uses a microfluidic device to detect MRD and searches unprocessed peripheral blood for circulating leukemic cells (CLCs). Antibodies immobilized within three separate microfluidic devices affinity-selected CLC subpopulations expressing CD33, CD34, and CD117 cell surface antigens commonly expressed by AML leukemic cells so that each subpopulation?s CLC numbers could be tracked to determine the onset of relapse. Staining against aberrant markers (e.g. CD7, CD56) identified low levels (11- 2,684/mL) of CLCs. Using our device to follow CLCs from AML patients after a stem cell transplantation (SCT), we detected MRD at an earlier stage (~2 months earlier) compared to both MFC and PCR, which used bone marrow biopsies. This earlier detection of relapse in AML post-SCT can enable curative clinical decisions for AML patients. Due to the minimally invasive nature of the assay, it allowed for more frequent testing as well. In this SBIR Phase II proposal, we plan to expand and develop for commercialization our microfluidic assay for AML CLC analysis providing coverage for >95% of all AML cases. Given the strong data generated to date and the urgent diagnostic need for an improved commercially available easy-to-implement MRD assay for frequent monitoring, broad patient coverage and early detection of AML relapse we propose a Direct-to-Phase II work-plan toward the commercialization development of this powerful assay.